Oligodendrocyte (OL) injury results in loss of myelin, failure of myelination, axonal damage and ultimately cell death. We have shown that increased intracellular Ca++ mediates damage in mature membrane sheet-bearing OLs. We hypothesize that the duration, magnitude and source of increased intracellular Ca++ are major determinants of the nature and outcome of OL damage. Specifically, we will ask whether a given mode of increasing Ca++ leads to membrane sheet retraction, OL death or both. We will compare Ca++-mediated injury elicited by thapsigargin to that elicited by the NO donor SNAP and by the glutamate receptor agonist kainate. In parallel with experiments on enriched OL cultures, we will utilize a myelinating co-culture system to examine the effects of axons on the responses of OLs to Ca++-mediated injury. Our long term objective is to identify strategies for protecting OLs from damage and optimizing myelin repair in white matter injury associated with diseases such as multiple sclerosis or with the sequelae of stroke or trauma. Specific Aim 1 utilizes laser cytometry to define the changes in Ca++ initiated in mature OLs by thapsigargin, NO and kainate. A range of doses will be compared for their effects on Ca++, membrane sheet retraction, and OL viability. Specific Aim 2 analyzes changes in cytoskeleton and rates of endocytosis which accompany Ca++-mediated retraction of OL membrane sheets, with focus on endocytosis of WGA receptors and proteolipid protein. Specific Aim 3 analyzes the role of increased Ca++ in the pathways leading to necrotic or apoptotic cell death. The contribution of each pathway will be assessed with various doses of the agents. Specific Aim 4 analyzes changes in gene expression accompanying OL responses to increased intracellular Ca++. Changes in message levels for myelin proteins (MBP, PLP, and DDM-20), relevant transcription factors (SCIP, GtX, and CREB) and immediate early genes (zif , c-fos, and c-jun) will be measured by nuclease protection assay or Northern blotting. In Specific Aim 5, the effects of axons on OL responses to increased OL Ca++ will be examined. Myelinated DRG-OL cultures will be exposed to thapsigargin, NO, or kainate. Cultures will be grown in a two-compartment system, so that agents can be applied to the axon-myelin-glia compartment, but not the neuronal cell bodies. Effects on myelin, OL viability, and axonal integrity will be evaluated by immunocytochemical staining. In situ hybridization and RT-PCR will assess changes in message levels. In the last four specific aims, the initial steps in signaling pathways that ultimately lead to Ca++-mediated injury will be examined by assessing the effects of Ca++ chelation at various times, and by determining whether cyclic AMP or calpains are involved.